title: A fast, cost-efficient and sensitive approach for KRAS mutation detection using multiplexed primer extension with IP/RP-HPLC separation.
abstract: Mutations in the KRAS gene are very important diagnostic and prognostic markers in cancer. Particularly, KRAS mutations at codons 12 and 13 have a high prognostic value for EGFR-directed antibody therapies. Several methods are available to detect the most common mutations, some of them are commercialized. The most frequently used techniques, allele-specific PCR or direct sequencing, are not standardized and often lack sensitivity to detect low amounts of mutated tumor cells in paraffin-embedded tissue-blocks leading to a high number of false-negatives. Here we present a reliable, fast, cost-effective and sensitive approach for KRAS mutation detection that has a high potential for standardized large scale screening. The method is based on multiplexed primer extension reactions coupled to HPLC separation. The highly sensitive assay gives easily interpretable and reproducible results at affordable costs. We describe the method and an application example for diagnosis in early colorectal cancer screening.
authors.affiliation: Saarland University, FR 8.3 Life Sciences, Genetics/Epigenetics, 66041 Saarbrücken, Germany. s.tierling@mx.uni-saarland.de
authors.forename: Sascha
authors.initials: S
authors.lastname: Tierling
authors.forename: Christine
authors.initials: C
authors.lastname: Sers
authors.forename: Annika
authors.initials: A
authors.lastname: Lehmann
authors.forename: Jörn
authors.initials: J
authors.lastname: Walter
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D055088:Early Detection of Cancer; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins